Autoreactive T cells that are capable of inducing autoimmune diseases exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of regulatory T cells. We have demonstrated that the regulatory T cells can be easily identified in normal lymphoid tissues by co- expression of CD4 and the interleukin-2 receptor alpha chain (CD25). Transfer of CD4+ CD25- T cells to immunoincompetent mice results in the development of autoimmune disease that can be prevented by co-transfer of CD4+CD25+ cells. Our recent studies have focused on defining the mechanism of action of the CD4+CD25+ in vitro. CD4+CD25+ T cells are completely non-responsive to stimulation via their T cell receptor due to an inability to produce IL-2. When mixed with CD4+CD25- cells, they suppress proliferation by blocking transcription of the IL-2 gene in the CD25- population. Suppression is mediated by a T-T interaction. We have examined the contribution of both cytokines and the CD28-CTLA-4/CD80-CD86 pathway in the activation of CD25+ T cells. Using a two-step culture system in which the CD4+CD25+ cells are preactivated in the absence of CD4+CD25- T cells, we have demonstrated that IL-2 or IL-4, but not other cytokines, are needed together with the TCR stimulus to generate potent suppressor activity. Inhibition of the interaction of CD28 or CTLA-4 with CD80-/CD86 in the pre-culture did not prevent the induction of suppressor function, nor did inhibition of costimulatory signals inhibit the ability of fresh CD25+ T cells to inhibit CD8+ responders under conditions where activation of the responders was costimulation independent. Activation of CD25+ cells could also be readily achieved in the presence of antigen presenting cells from CD80/CD86 double deficient donors. These studies are most consistent with the view that activation of CD25+ T cells requires IL-2/IL-4 for their survival/differentiation into effector cells, but is completely independent of CD28/CTLA-4-mediated costimulation. We have employed microarray technology to identify several genes that encode factors that may be related to the suppressive function of CD4+CD25+ cells. Resting CD25+ T cells selectively express the glucocorticoid induced TNF receptor (GITR, TNFRSF18) and engagement of this molecule by antibody or by its ligand (GITR-L) abrogates suppression mediated by CD4+CD25+ T cells. We have generated monoclonal antibodies to the GITR-L and shown that it has a unique tissue distribution with expression being limited to resting B cells and dendritic cells. Inflammatory signals appear to down-regulate GITR-L expression. Manipulation of GITR/GITR-L interactions on both CD4+CD25+ and CD4+CD25- T cells interactions may result in enhancement or inhibition of regulatory T cell function. In addition to autoimmune disease, regulatory T cells also play an important role in the persistence of chronic infectious diseases. We have shown that the persistence of Leishmania major infection in resistant C57BL/6 mice is controlled by CD4+CD25+ T cells. In mice that lack regulatory T cells, L. major infection is completely cleared, but these mice fail to develop immunity to the parasite. Thus, CD4+CD25+ T cells have pleiotropic effects on immune responses in autoimmunity, tumor immunity, and infectious disease. Manipulation of regulatory T cell function should represent a novel adjunct to the therapy of several diseases.